The present invention relates to pyrrolobenzodiazepines (PBDs), and is particularly concerned with the use of these compounds as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT), gene-directed enzyme-prodrug therapy (GDEPT), photodynamic therapy (PDT) and naturally present enzyme-prodrug therapy (NPEPT).
Pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the most preferred sequence is PuGPu (Purine-Guanine-Purine). The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber et al., 1965 J. Am. Chem. Soc., 87, 5793-5795; Leimgruber et al., 1965 J. Am. Chem. Soc., 87, 5791-5793). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston et al., 1994 Chem. Rev. 1994, 433-465). Family members include abbeymycin (Hochlowski et al., 1987 J. Antibiotics, 40, 145-148), chicamycin (Konishi et al., 1984 J. Antibiotics, 37, 200-206), DC-81 (Japanese Patent 58-180 487; Thurston et al., 1990, Chem. Brit., 26, 767-772; Bose et al., 1992 Tetrahedron, 48, 751-758), mazethramycin (Kuminoto et al., 1980 J. Antibiotics, 33, 665-667), neothramycins A and B (Takeuchi et al., 1976 J. Antibiotics, 29, 93-96), porothramycin (Tsunakawa et al., 1988 J. Antibiotics, 41, 1366-1373), prothracarcin (Shimizu et al, 1982 J. Antibiotics, 29, 2492-2503; Langley and Thurston, 1987 J. Org. Chem., 52, 91-97), sibanomicin (DC-102)(Hara et al., 1988 J. Antibiotics, 41, 702-704; Itoh et al., 1988 J. Antibiotics, 41, 1281-1284), sibiromycin (Leber et al., 1988 J. Am. Chem. Soc., 110, 2992-2993) and tomamycin (Arima et al., 1972 J. Antibiotics, 25, 437-444). PBDs are of the general structure: 
They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (Nxe2x95x90C), a carbinolamine (NHxe2x80x94CH(OH)) or a carbinolamine methyl ether (NHxe2x80x94CH(OMe))at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, 1975 In Antibiotics III. Springer-Verlag, New York, pp. 3-11; Hurley and Needham-VanDevanter, 1986 Acc. Chem. Res., 19, 230-237). Their ability to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents.
The use of prodrugs represents a very valuable clinical concept in cancer therapy. For example, a prodrug may be converted into an antitumour agent under the influence of an enzyme that is linked to a monoclonal antibody so that it can bind to a tumour associated antigen. The combination of such a prodrug with such an enzyme monoclonal antibody conjugate represents a very powerful therapeutic strategy. This approach to cancer therapy, often referred to as xe2x80x9cantibody directed enzyme/prodrug therapyxe2x80x9d (ADEPT) is disclosed in WO88/07378.
A further therapeutic approach termed xe2x80x9cvirus-directed enzyme prodrug therapyxe2x80x9d (VDEPT) has been proposed as a method for treating tumour cells in patients using prodrugs. Tumour cells are targeted with a viral vector carrying a gene encoding an enzyme capable of activating a prodrug. The gene may be transcriptionally regulated by tissue specific promoter or enhancer sequences. The viral vector enters tumour cells and expresses the enzyme, thereby converting the prodrug into the active drug within the tumour cells (Huber et al., Proc. Natl. Acad. Sci. USA (1991) 88, 8039). Alternatively, non-viral methods for the delivery of genes have been used. Such methods include calcium phosphate co-precipitation, microinjection, liposomes, direct DNA uptake, and receptor-mediated DNA transfer. These are reviewed in Morgan and French, Annu. Rev. Biochem., 1993, 62;191. The term xe2x80x9cGDEPTxe2x80x9d (gene-directed enzyme prodrug therapy) is used to include both viral and non-viral delivery systems.
Photodynamic therapy (PDT) provides another method which uses prodrugs to deliver desired drugs to specific sites in the human body. Advances in the field of light delivery to internal areas of the body allow delivery to organs and other areas without the need for any extensive surgical procedures. The activation process can be extremely site specific, as the direction of a laser beam can be controlled with great precision, and the beam diameter can be reduced far below that of a single cell, minimising any possible damage to other neighbouring tissue from unwanted activation of the drug. The high energy of ultra-violet light (e.g. 350 nm equivalent to 340 kJ/mol) is sufficient to break a range of chemical bonds, since the bond energy spectrum of the majority of organic molecules lies between 250 and 420 kJ/mol. For example, there has been wide application of the photochemical deprotection of amino acids, peptides and polysaccharides from their o-nitrobenzyl carbamate, CBZ, and 4,5-dimethoxy-2-nitrobenzyl carbamate forms at wavelengths longer than 350 nm.
A further class of prodrugs is those where the protecting group is removed by an enzyme naturally present at the desired site of action. These enzymes include dopa-decarboxylase, L-xcex3-glutamyl transpeptidase, and mixed function oxidases and reductase (e.g. DT-diaphrase). This is method termed xe2x80x9cnaturally present enzyme-prodrug therapy (NPEPT) in this application. One enzyme of particular interest is glutathione transferase (GST), which forms part of a major cellular defence mechanism based on the use of the tripeptide, glutathione, as a scavenger of toxic electrophiles. GST acts as a catalyst in the reaction between glutathione and its target electrophiles. A consequence of this defence mechanism is the inactivation of electrophilic therapeutic agents. Many human tumour cells exhibit elevated GST levels compared to normal cells and the association of GST with resistance to DNA alkylating agents has been demonstrated by Lewis et al. (Carcinogenesis 1988, 9, 1283-1287), Kuzmich et al. (Journal of Biochemistry 1992, 281, 219-224), and Tew et al. (Glutathione-S-transferase and anti-cancer drug resistance, in Mechanism of Drug Resistance in Neoplastic Cells; Wooley, P. V., Tew, Kr. D., Eds.; Academic Press: Orlando, Fla., 1987; pp141-159). Chemotherapeutic agents that take advantage of this intrinsic property of cancer cells may prove highly useful in treating refractory cancers.
Prodrugs which make use of this elevated GST level have been made (Satyam et al., Med. Chem. 1996, 39, 1736-1747). They have a glutathione molecule linked via a 2-sulphonylethyloxycarbonyl linker to a phosphorodiamidate mustard. An alternative type of prodrug has made use of the closely related 2-phenylsulphonylethyloxycarbonyl (Psec) group (Nicolaou et al., Science, 1992, 256, 1172-1178). Such prodrugs showed selectivity between healthy human bone marrow cells and promeocytic and T cell leukemia tumour lines.
A first aspect of the present invention provides a compound with the formula I: 
wherein:
R10 is a therapeutically removable nitrogen protecting group;
R2 and R3 are independently selected from: H, R, OH, OR, xe2x95x90O, xe2x95x90CHxe2x80x94R, xe2x95x90CH2, CH2xe2x80x94CO2R, CH2xe2x80x94CO2H, CH2xe2x80x94SO2R, Oxe2x80x94SO2xe2x80x94R, CO2R, COR and CN;
R6, R7 and R9 are independently selected from H, R, OH, OR, halo, amino, nitro, Me3Sn; or R, and R, together form a group xe2x80x94Oxe2x80x94(CH2)pxe2x80x94Oxe2x80x94, where p is 1 or 2;
X is S, O or NH;
R11 is either H or R;
where R is a lower alkyl group having 1 to 10 carbon atoms, or an aralkyl group (i.e. an alkyl group with one or more aryl substituents), preferably of up to 12 carbon atoms, whereof the alkyl group optionally contains one or more carbon-carbon double or triple bonds, which may form part of a conjugated system, or an aryl group, preferably of up to 12 carbon atoms; and is optionally substituted by one or more halo, hydroxy, amino, or nitro groups, and optionally contains one or more hetero atoms, which may form part of, or be, a functional group;
and where there is optionally a double bond between C1 and C2 or C2 and C3;
and R8 is selected from H, R, OH, OR, halo, amino, nitro, Me3Sn, where R is as defined above, or the compound is a dimer with each monomer being the same or different and being of formula I, where the R8 groups of the monomers form together a bridge having the formula xe2x80x94Txe2x80x94Rxe2x80x2xe2x80x94Txe2x80x94 linking the monomers, where Rxe2x80x2 is an alkylene chain containing from 3 to 12 carbon atoms, which chain may be interrupted by one or more hetero atoms and/or aromatic rings, e.g. benzene or pyridine, and may contain one or more carbon-carbon double or triple bonds, and each T is independently selected from O, S or N.
If R is an aryl group, and contains a hetero atom, then R is a heterocyclic group. If R is an alkyl chain, and contains a hetero atom, the hetero atom may be located anywhere in the alkyl chain, e.g. xe2x80x94Oxe2x80x94C2H5, xe2x80x94CH2xe2x80x94Sxe2x80x94CH3, or may form part of, or be, a functional group, e.g. carbonyl, hydroxy.
R is preferably independently selected from a lower alkyl group having 1 to 10 carbon atoms, or an aralkyl group, preferably of up to 12 carbon atoms, or an aryl group, preferably of up to 12 carbon atoms, optionally substituted by one or more halo, hydroxy, amino, or nitro groups. It is more preferred that R groups are independently selected from a lower alkyl group having 1 to 10 carbon atoms optionally substituted by one or more halo, hydroxy, amino, or nitro groups. It is particularly preferred that R groups are unsubstituted straight or branched chain alkyl groups, having 1 to 10, preferably 1 to 6, and more preferably 1 to 4, carbon atoms, e.g. methyl, ethyl, propyl, butyl.
Alternatively, R6, R7, R8, and R9 may preferably be independently selected from R groups with the following structural characteristics:
(i) an optionally substituted phenyl group;
(ii) an optionally substituted ethenyl group;
(iii) an ethenyl group conjugated to an electron sink.
The term xe2x80x98electron sinkxe2x80x99 means a moiety covalently attached to a compound which is capable of reducing electron density in other parts of the compound. Examples of electron sinks include cyano, carbonyl and ester groups.
The term xe2x80x98therapeutically removable nitrogen protecting groupxe2x80x99 means any group which can protect the 10-nitrogen, but which is removable under therapeutic conditions in vivo, that is, removable under conditions which occur or can be caused to occur in vivo and are medically acceptable generally by elimination to produce a N10-C11 imine group or an equivalent, capable of interacting with DNA. The removal of the protecting group should leave the rest of the structure of the PBD unaffected.
Suitable removal techniques include applying light, e.g. with a wavelength of 250 to 400, or 550 nm, changing the ambient pH, or cleavage by the action of an enzyme. One particularly suitable enzyme is nitroreductase, although other suitable enzymes include penicillin V/G amidase, P-lactamase, phosphatase, L-xcex3-glutamyl transpeptidase, and xcex1-galactosidase. The action of some of these enzymes is described in Jungheim, L. N. and Shepherd, T. A., Design of Antitumour Prodrugs: Substrates for Antibody Targeted Enzymes, Am. Chem. Soc. Chem. Rev., 1994, 94: 6, 1553-1566. Another particularly suitable enzyme is glutathionare transferase, as discussed above.
One possible group is R10 of the formula II: 
wherein n is 0 to 3, R(I) is H or R, and R(II) is one or more optional substituents independently selected from NO2, OR, or R, where R is as defined in any of the definitions above; and if two substituents R(II) are on adjacent atoms, they may together be of the formula xe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94, where m is 1 or 2. R(II) is preferably NO2.
If the therapeutically removable group R10 is one which is susceptible to nitroreductase, it may be of the formula III: 
wherein n is 0 to 3, R(I) is H or R, and R(III) is one or more optional substituents independently selected from NO2, OR or R, where R is as defined in any of the definitions above, and if two substituents R(III) are on adjacent atoms, they may together be of the formula xe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94, where m is 1 or 2.
Another possible therapeutically removable nitrogen protecting group, R10, is of the formula XI: 
where R is as defined in any of the definitions above and n is 0 to 3, preferably 0. For this formula, R is most preferably a phenyl group, substituted or unsubstituted. This protecting group may be removable by the action of glutathione transferase (GST), which is present at high levels in many human tumour cells (see above).
It is preferred in compounds of formula I that X is O and, independently, that R11 is H.
If there is a double bond in the C ring, it is preferably between C2 and C3.
Additionally, it is preferred that R6 and R9 are H, and further preferred that R7 and R8 are independently selected from H, OH, and OR. It is further preferred that R2 and R3 are H.
If the compound of formula I is a dimer, the dimer bridge may be of the formula xe2x80x94Oxe2x80x94(CH2)pxe2x80x2xe2x80x94Oxe2x80x94, where pxe2x80x2 is from 1 to 12, preferably 3 to 9. Further, R6 and R9 are preferably H, and R7 is preferably independently selected from H, OH, and OR.
A second aspect of the present invention provides a method of preparing a compound of formula I as described in the first aspect of the invention wherein XR11xe2x89xa0OH, from a corresponding compound Ia which is a compound of formula I in which XR11=OH. A product in which XR11 is OR may be prepared by direct etherification of compound Ia. A product in which X is S may be prepared by treatment of compound Ia with R11SH and a catalyst (generally a Lewis acid such as Al2O3). A product in which X is NH may be prepared by treatment of compound Ia with an amine R11NH and a catalyst (generally a Lewis acid such as Al2O3).
A third aspect of the present invention provides a method of preparing a compound of formula Ia as described in the second aspect of the invention, by the oxidation of a compound of formula IVa: 
wherein the substituents of the compound of formula IVa are the same as for the compound of formula Ia to be prepared. (For preparation of dimeric compounds, the monomers linked through C8 by xe2x80x94Txe2x80x94Rxe2x80x2xe2x80x94Txe2x80x94 are both of formula IVa. Similar comments apply to other intermediates in dimer synthesis.) The preferred oxidation method is Swern oxidation.
A fourth aspect of the present invention provides a method of preparing a compound of formula IVa as described in the third aspect of the invention, by reacting a compound of formula Va: 
with a compound of formula VI:
Yxe2x80x94R10xe2x80x83xe2x80x83(VI)
wherein the substituents of the compounds of formulae Va and VI are the same as for the compound of formula IVa to be prepared, and Y is a halogen atom.
If the therapeutically removable nitrogen protecting group is to be of formula II, then it is preferred that the compound of formula VI is a haloformate of the formula VIa: 
wherein the substituents are as defined for the group of formula II, and Y is a halogen atom.
If the therapeutically removable nitrogen group is to be of formula XI, then it is preferred that the compound of formula VI is a haloformate of the formula VIb: 
where Y is a halogen atom, and R and n are as defined for formula XI.
A fifth aspect of the present invention provides an alternative synthesis of a compound of formula IVa as described in the third aspect of the invention, by reacting a compound of formula VII: 
with a compound of formula VI:
Yxe2x80x94R10xe2x80x83xe2x80x83(VI)
to form a compound of formula VIII: 
and then reacting the compound of formula VIII with a compound of formula IXa: 
(e.g. by means of (COCl)2), wherein the substituents for compounds of formulae VI, VII, VIII and IXa are the same as for the compound of formula IVa to be prepared, where Y is a halogen atom.
A sixth aspect of the present invention provides a method of preparing a compound of formula Ia as described in the second aspect of the invention, by the unmasking of a compound of formula IVb: 
wherein the substituents of the compound of formula IVb are the same as for the compound of formula Ia to be prepared, and Q is either S or O and R(IV) are independently selected from Me or Et or may together form xe2x80x94(CH2)qxe2x80x94 where q is 2 or 3. (For preparation of dimeric compounds, the monomers linked through C8 by xe2x80x94Txe2x80x94Rxe2x80x2xe2x80x94Txe2x80x94 are both of formula IVb. Similar comments apply to other intermediates in dimer synthesis.) The preferred unmasking method when Qxe2x95x90S is mercury-mediated unmasking. Unmasking when Qxe2x95x90O is preferably carried out by the use of acid conditions, e.g. TFA, methanol and water or palladium catalysis.
A seventh aspect of the present invention provides a method of preparing a compound of formula IVb as described in the sixth aspect of the invention, by reacting a compound of formula Vb: 
with a compound of formula VI:
Yxe2x80x94R10xe2x80x83xe2x80x83(VI)
wherein the substituents of the compounds of formulae Vb and VI are the same as for the compound of formula IVb to be prepared, and Y is a halogen atom.
If the therapeutically removable nitrogen protecting group is to be of formula II, then it is preferred that the compound of formula VI is a haloformate of the formula VIa: 
wherein the substituents are as defined for the group of formula II, and Y is a halogen atom.
An eighth aspect of the present invention provides an alternative synthesis of a compound of formula IVb as described in the second aspect of the invention, by reacting a compound of formula VII: 
with a compound of formula VI:
Yxe2x80x94R10xe2x80x83xe2x80x83(VI)
to form a compound of formula VIII: 
and then reacting the compound of formula VIII with a compound of formula IXb: 
(e.g. by means of (COCl)2), wherein the substituents for compounds of formulae VI, VII, VIII and IXb are the same as for the compound of formula IVb to be prepared, where Y is a halogen atom.
A ninth aspect of the present invention provides a method of making a compound of formula X: 
by cleavage of the therapeutically removable protecting group R10 of a compound of formula I as described in the first aspect of the invention, wherein the substituent groups of the compound of formula X are the same as the substituent groups of the compound I used.
A tenth aspect of the present invention provides a use of a compound of formula I, wherein the therapeutically removable nitrogen protecting group (R10)is enzyme labile, in conjunction with an appropriate enzyme in methods of ADEPT or GDEPT therapy. If the enzyme labile group is susceptible to nitroreductase, then compounds of formula I, may be used in conjunction with nitroreductase enzymes (for example, those isolated from E. coli) in methods of ADEPT and GDEPT therapy.
An eleventh aspect of the present invention provides a use of a compound of formula I, wherein the therapeutically removable nitrogen protecting group (R10)is photolabile, in conjunction with light of wavelengths between 250 and 400 or 550 nm in methods of PDT.
A twelfth aspect of the invention provides a use for a compound of formula I, where the therapeutically removable nitrogen protecting group (R10) is labile by conditions occurring naturally at specific localised sites in the patient in therapy. Suitable compounds of formula I may be those susceptible to a nitroreductase enzyme when used to treat hypoxic tumour cells, or those susceptible to enzymes which are naturally occurring at specific localised sites, such as glutathione transferase.
The drug produced by the cleavage of the therapeutically removable nitrogen protecting group, in either the tenth or eleventh or twelfth aspect of the invention, may be used for treating cancers or other site-specific diseases where a local increase of toxicity is beneficial to the patient. Cancers that may be treated are solid cancers including ovarian, colonic cancer, renal, breast and bowel CNS, melanoma, as well as leukemias. Such drugs may also be suitable for treating bacterial, viral or parasitic infections by exploiting a unique enzyme produced at the site of the infection which is not natural to the host, or by exploiting an elevation in the amount of an enzyme which does naturally occur in the host.
A thirteenth aspect of the present invention is a pharmaceutical composition comprising a compound of formula I as described in the first aspect of the invention. Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a compound of formula I, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Capsules may comprise a solid carrier such as gelatin.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Solution, or Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
A fourteenth aspect of the present invention provides the use of a compound of formula I as described in the first aspect of the invention, to prepare a medicament for the treatment of neoplastic disease or other site-specific diseases where a local increase of toxicity is beneficial to the patient. The compound of formula I may be provided together with a pharmaceutically acceptable carrier or diluent. The preparation of a medicament is described in relation to the thirteenth aspect of the invention.